Polyelectrolyte Flocculation as an Aid to Recovery of Enteroviruses from Oysters

A simple and rapid method for recovering enteroviruses from oysters is described. A polycation sewage flocculant promoted cohesion of oyster solids and thereby aided separation of these from the viruses. Recovery of 80 to 100% of experimentally inoculated virus was achieved, and the suspension or extract obtained could be inoculated directly into tissue cultures or concentrated first for greater sensitivity.     

Minicultures of Mammalian Cells in a New Plastic Plate

A new disposable micro tissue culture plate was developed and tested for use in virological procedures. Miniature mammalian cell cultures (minicultures) were grown in these plates. Each plate contained 96 circular cultures in flat wells (7 mm in diameter). Replicate titrations of a number of viruses were performed in various tissues. Excellent reproducibility was demonstrated. Mean infectivity titers determined by miniculture methods were generally within 0.6 log₁₀/ml of macro tube titrations. Standard tissue culture assay techniques such as hemadsorption, interference titration, and microneutralization were easily carried out with this method and were very reproducible. Development of this noncytotoxic disposable micro tissue culture plate now permits the routine performance of rapid, reliable, and reproducible tissue culture tests at a very significant reduction in cost and labor.     

Conditionally Reprogrammed Human Normal Airway Epithelial Cells at ALI: A Physiological Model for Emerging Viruses

Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models(ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells(CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming(CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid(2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for airliquid interface(ALI) polarized 3 D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses.     

Selected laboratory aspects of influenza surveillance

The importance of virologically documented infections in influenza surveillance is well recognized and has been reaffirmed in recent reviews. The large number of specimens tested in surveillance make efficiency and low cost of virologic methods important. Based on observations made by others and our work with reisolation of stored specimens we have used the continuous line tissue cultures MDCK and LLC-MK2 for virus isolation in large-scale influenza surveillance studies for three years. Both cell lines were equally successful in detecting influenza A viruses in 77 fresh, virus-positive specimens. However, during the influenza B outbreak of 1979--80, of 473 specimens positive in either or both tissue cultures, 54 were positive only in MDCK and just six in LLC-MK2 only. For parainfluenza viruses, LLC-MK2 was much superior to MDCK. The most promising alternative to tissue culture at this time, based on a review of the literature, appears to be enzyme immunoassay. Sensitivity sufficient for direct detection of viral antigen in routine specimens currently requires fluorescent or radioactive substrates. Identification of early virus growth in continuous cell line cultures by enzyme immunoassay is practical now and can be considered.     

Safe biotechnology (5). Recommendations for safe work with animal and human cell cultures concerning potential human pathogens

The benefits of using animal or human cell cultures have been clearly demonstrated in diagnostic and therapeutic research and in their application for manufacturing. Cell cultures serve as a tools for the production of vaccines, receptors, enzymes, monoclonal antibodies and recombinant DNA-derived proteins. They represent an integral part of drug development for which corresponding facilities, equipment and manufacturing processes are required. Although the cells themselves offer no particular risk to workers in laboratories and production areas or to the environment, the cell cultures may be contaminated with viruses, mycoplasma, bacteria, yeast and fungi or might contain endogenous viruses. The containment level for animal and human cells is therefore determined by the risk class of these agents. The history of animal and human cell cultures has proved that they can be handled safely. The recommendations in this publication concern the safe handling of cell cultures (tissue explants, primary cell cultures) and permanent cell lines of animal and human origin. A classification system of safety precautions has been elaborated according to the potential for contamination with the pathogenic agents involved. Correspondence to: DECHEMA, EFB Secretariate, Postfach 150101, W-6000 Frankfurt/Main 15     

The antiviral action of a modified bacterial ribonuclease]

The antiviral activity of a bacterial ribonuclease conjugate with chitosane of Kamchatka crab (in a form of water soluble chito-oligosaccharides) has been studied. The conjugate inhibitory activity for A and B viruses as well as to Sindbis arbovirus in tissue cultures is shown. The preparation efficiency at intramuscular and intranasal administration was observed at experimental influenza infection of white mice.     

Rapid Detection and Identification of Respiratory Viruses by Direct Immunofluorescence

The use of fluorescein-conjugated antiserum against respiratory syncytial (RS) and parainfluenza 1 and 3 viruses was compared with conventional techniques in the rapid detection of virus in tissue cultures inoculated with pharyngeal specimens known to contain these viruses. Twenty-three specimens were tested: 9 RS, 8 parainfluenza 1, and 6 parainfluenza 3. The fluorescent-antibody technique (FA) detected virus in 52% of the tissue cultures in 24 hr, and, by 72 hr, 22 of the 23 cultures were FA-positive whereas only 5 were positive by conventional techniques. Additionally, conjugated antisera were prepared against herpes simplex, influenza A₂, and adenovirus type 5. All conjugates stained only the homologous virus and were 100- to 10,000-fold more sensitive than conventional techniques in detecting descending dilutions of virus inocula by 24 hr. With the procedures described, several antisera could be conjugated and ready for use within 24 hr. Serum fractionation was by ammonium sulfate precipitation, and with the procedure outlined virtually complete recovery of the globulin fraction and elimination of all of the albumin were accomplished.     

Murine virus susceptibility of cell cultures of mouse, rat, hamster, monkey, and human origin.

Studies were initiated to determine the practicality of using various tissue cultures for the propagation of murine viruses isolated from laboratory animals. The cytopathogenic effects of 10 murine viruses known to cause disease in laboratory rodents were compared in monolayer cultures of L929, BHK-21, WI-38, BSC-1, and Vero cells. The susceptibility of primary hamster embryo, hamster kidney, mouse embryo, mouse kidney, and rat embryo cell cultures was also tested. Seven of the viruses produced effects in at least 1 of the cell substrates. The remaining 3 viruses, namely H-1, K, and mouse hepatitis, produced no effects in the cell cultures tested.     

Simian immunodeficiency virus variants that differ in pathogenicity differ in fitness under rapid cell turnover conditions

Simian immunodeficiency virus (SIV) has been shown to progress through a number of changes that lead to the emergence of pathogenic viral variants in macaques initially infected with a mildly cytopathic variant, SIVMneCL8. One of these late-stage isolates, SIVMne170, replicates to high levels in vivo and causes a rapid disease course when reintroduced into na?ve macaques, resulting in a viral set point up to 3,000-fold higher than the set point of the parental virus, SIVMneCL8. However, in cell culture both viruses replicate with similar kinetics. One major difference between in vivo and in vitro cultures is the life span of the infected cells. Here, we manipulated the life span of infected cells in vitro, and we show that the fitness of SIVMne170 in cultures with a limited cell life span dramatically increased compared to its fitness in cultures with a nonlimited life span of cells. The increase in fitness was at least partially due to the fact that the rapid turnover system eliminates the negative influence of the cytopathic effects associated with replication of SIVMne170. Because the relative fitness of SIVMneCL8 and SIVMne170 observed in the rapid turnover system more accurately reflects their fitness in vivo, the system represents an improved approach to comparing relative fitness of viruses.     

A method for the rapid production of fine plant cell suspension cultures

A method has been devised which allows the rapid production of fine suspension cultures of small aggregate size from suspension cultures of large average aggregate size, such as those of Capsicum frutescens. The method, which uses a Waring blender for aseptic homogenisation of cultures, has also been shown to be effective in rapidly producing suspension cultures from callus cultures. The suspension cultures so produced are particularly useful for immobilisation, such as in porous polyurethane foam matrices.     

Microbial hazards in plant tissue and cell cultures

Summary A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.     

Response of Children to Inactivated Measles Vaccine Prepared in Bovine Renal Cell Culture

Formalin-inactivated, alum-adsorbed measles vaccine was readily prepared from virus grown in calf kidney cell culture infected with the Sugiyama strain of measles virus which had been adapted to this cell culture. The vaccine induced no side reaction of any consequence in vaccinated children, but demonstrated antigenic capacity in children as well as guinea pigs, comparable to that of currently used killed measles vaccines prepared from virus grown in monkey kidney or chick embryo tissue cultures. The host system employed for the preparation of this vaccine has an advantage over monkey kidney or chick embryo tissue cultures which are currently used for manufacture of killed measles vaccine. Bovine kidneys are much easier to obtain and cultivate. Of importance is the fact that calf kidneys are practically free of latent virus, whereas monkey kidneys and chick embryos frequently harbor latent viruses.     

Foreign protein production using plant cell and organ cultures: Advantages and limitations

Plants and plant tissue cultures are used as host systems for expression of foreign proteins including antibodies, vaccines and other therapeutic agents. Recombinant or stably transformed plants and plant cell cultures have been applied for foreign protein production for about 20 years. Because the product concentration achieved exerts a major influence on process economics, considerable efforts have been made by commercial and academic research groups to improve foreign protein expression levels. However, post-synthesis product losses due to protease activity within plant tissues and/or extracellular protein adsorption in plant cell cultures can negate the benefits of molecular or genetic enhancement of protein expression. Transient expression of foreign proteins using plant viral vectors is also a practical approach for producing foreign proteins in plants. Adaptation of this technology is required to allow infection and propagation of engineered viruses in plant tissue cultures for transient protein expression in vitro.     

Rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.     

Technique for cultivation of transitional epithelium from mammalian urinary bladder.

A technique for initiating cultures of epithelial (urothelial) cells from mammalian urinary bladder has been described. Urothelial cells obtained by this method have been used to support replication of viruses and as controls for immunological, biochemical, chromosome, and electron microscopy studies. Both light and electron microscopic studies of cultured cells suggest that they are epithelial and not a mixed culture.     

The origin and characteristics of a pig kidney cell strain,LLC-PK1

Summary A stable epithelial-like pig kidney cell strain has been established. This strain has been carried through more than 300 serial passages, has remained free of microbial and viral contaminants, and has retained a near diploid number of chromosomes. Attempts to produce tumors with these cells in immunosuppressed laboratory animals have been uniformly negative. The cells have grown rapidly in monolayer cultures with a split ratio of 1 to 15 at weekly intervals, but have failed to proliferate in suspension cultures. A subline adapted to growth on serum-free medium 199 has been carried through 145 passages on this medium. Several unusual morphologic features have been observed in these cultures including three-dimensional “domelike” structures. These cells have been found susceptible to some viruses and have been especially useful for viruses of domestic animals. LLC-PK₁ cells have produced significant levels of plasminogen activator. Histopathological examinations of animal tissues were done by Dr. C. G. Culbertson. Tests for mycoplasma were performed by Mr. D. H. Holmes.     

Simultaneous quantification of eudesmanolides and thymol derivatives from tissues of Inula helenium and I. royleana by reversed-phase high-performance liquid chromatography

A simple and rapid isocratic reversed-phase high-performance liquid chromatographic method for the quantification of alantolactone/isoalantolactone and three thymol derivatives in roots and root cultures of Inula helenium and I. royleana has been developed. The method could be applied to screen raw materials in search for highly productive plants and in vitro cultures.     

Use of the DNA-specific fluorochrome olivomycin for work with cell cultures]

A high-sensitive, easy and rapid proximate method has been developed to reveal mycoplasmas in the monolayer cell cultures using home fluorescent antibiotic olivomycin. This method has been used to screen many cell lines with its high effectiveness being shown. As based on this method the following control methods are worked out: indication of mycoplasmas in animal blood sera, used for the cultivation of cell lines detection of contaminant microorganisms of the cell cultures (fungi and bacteria) and human ureaplasmas.